Analysis of MCMV m154.

<p>(A) The deduced amino acid sequence of m154 is shown, with the predicted leader peptide in yellow and the transmembrane region in blue. The putative N-linked glycosylation site (underlined) and O-glycosylation sites (bold) are indicated. (B) Peritoneal macrophages were mock-infected (mock) or infected with wt MCMV at an moi of 10 in the absence (lane -) or presence of cycloheximide and actinomycin D (lane CHX), or phosphonoacetic acid (lane PPA). Cell lysates were prepared at 72 hpi, except for the cycloheximide treated samples that were collected 16 hpi, separated under reducing conditions by SDS-PAGE (10%), and transferred to a nitrocellulose membrane. The blot was proved with the anti-m154 mAb or anti-MCMV IE1 (Croma 101) mAb followed by anti-mouse IgG HRP. A mAb anti-β-actin followed by an anti-rabbit IgG HRP was used as an internal control. (C) Peritoneal macrophages, either mock-infected or infected with wt MCMV at an moi of 10 for 72 h, were fixed, permeabilized, and stained with anti-m154 mAb followed by an anti-mouse IgG Alexa fluor 555. Nuclei were stained with the DAPI reagent. The cells were examined under a microscope at 405 nm (DAPI, panels a and d) and at 555–565 nm (Alexa-555, panels b and e). The overlaid images are shown in panels c and f. (×10 magnification). (D) Peritoneal macrophages, either mock-infected (mock) or infected with wt MCMV at an moi of 10, were surface-labeled with biotin. Cell lysates were prepared at 72 hpi and subjected to immunoprecipitation with anti-m154 mAb, followed by SDS-PAGE (10%) separation and Western blot analysis as indicated in B, using streptavidin-POD conjugate. Molecular weights in kilodaltons are indicated. (E) Flow cytometry analysis of 300.19 cells stably transfected with empty pDisplay vector expressing HA (left panel) or the construct HA-m154 (right panel) and stained after 24 h with biotin anti-HA mAb (black line) or isotype control (dashed line) followed by streptavidin-PE. (F) 300.19 cells non-transfected (shaded histogram) or stably transfected with the construct HA-m154 (black line histogram) were analyzed by flow cytometry for surface expression of CD48 as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004000#ppat-1004000-g001" target="_blank">Figure 1</a>. The corresponding isotype was used as a negative control (dashed line histogram).